The dynamics of secretory vesicles in living hyphae of the pathogen Ustilago maydis.

丝状真菌是一种进化成功的生物，作为菌根与植物相互作用的共生体和植物碎片的分解者，具有巨大的生态重要性。它们用于蛋白质的工业生产，并作为病原体对公共卫生和农业构成威胁。丝状真菌的基本单位是菌丝；这通常由一串细长的细胞组成，这些细胞通过尖端的扩张生长，这个过程被称为尖端生长。这种生长模式允许侵入组织和基质。菌丝尖端的生长需要不断地向菌丝尖端提供新合成的膜、蛋白质和细胞壁前体，这一过程被称为分泌。这些物质的载体是分泌性运输囊泡，它们被带到尖端，在那里它们与质膜融合。在本资助申请的背景下，重要的是要注意到，我们目前对分泌物的看法是，高尔基后囊泡单向移动到菌丝尖端，在那里它们与质膜融合，从而传递膜和蛋白质。分泌囊泡被运送到菌丝尖端的机制尚不清楚。与其他极化细胞（如动物神经元）类似，假设微管和运动蛋白马达介导分泌囊泡的远距离运输，而质膜上的短程运动由另一个系统介导，由肌球蛋白和丝状肌动蛋白（F-actin）组成。事实上，最近对许多丝状真菌的研究，包括植物病原体黑穗病菌，表明微管，动力蛋白，f -肌动蛋白和肌球蛋白是菌丝生长所必需的。然而，从这些数据得出的大多数结论都是推测性的。这是由于分泌货物的运动从未可视化，也没有对细胞中所有运动的系统研究进行过。在这个项目中，我们将应对这一挑战。我们已经开发了一种显微镜装置，使我们能够可视化单个几丁质合成酶囊泡的运动。我们将标记许多其他分泌蛋白，包括分泌酶，并将监测它们的输送到生长的菌丝尖端。在使用红色和绿色荧光蛋白的共定位研究中，我们将确定货物是在相同还是不同的运输囊泡中行进，从而阐明分泌途径。我们将确定构成囊泡运动基础的细胞骨架元素，并通过利用现有的突变蛋白结构来解决所有运动蛋白和肌球蛋白在分泌中的作用。最后，我们将进一步探讨壳体双向运动的原因。这种行为是出乎意料的，我们将研究它是否是分泌囊的一般特征。随后，我们将使用光激活荧光蛋白来表征这种运动性，以深入了解这种现象的原因。总之，我们将结合分子遗传学和生命细胞成像：(1)确定菌丝细胞的分泌途径，(2)解决10种动力蛋白和4种肌球蛋白在分泌囊泡运动中的作用，(3)表征分泌囊泡的双向运动，以便理解这种行为背后的逻辑。该项目的预期结果将是对丝状真菌分泌途径的新见解。我们将全面了解货物到达菌丝尖端的途径，以及运动蛋白和肌凝蛋白在分泌囊泡递送中的作用。如果发现分泌囊的双向运动是一个普遍的特征，那么目前的分泌模式将不得不被修改。因此，该项目将对真菌研究的各个方面产生根本性的兴趣，但在了解真菌致病性和重组蛋白的工业生产方面尤为重要。

Filamentous fungi are an evolutionarily successful group of organisms of enormous ecological importance as symbionts in mycorrhizal interactions with plants and decomposer of plant debris. They serve in industrial production of proteins and as pathogens pose a threat to public health and agriculture. The basic unit of a filamentous fungus is the hypha; this usually consists of a chain of elongated cells that grow by expansion at the tip, a process called tip growth. This mode of growth allows the invasion of tissue and substrate. Tip growth requires a continuous supply of newly synthesised membranes, proteins and cell wall precursors to the hyphal tip in a process named secretion. The carriers for these supplies are secretory transport vesicles that are taken to the tip where they fuse with the plasma membrane. It is important in the context of this grant application to note that our current view of secretion is that post-Golgi vesicles travel uni-directionally to the hyphal tip where they fuse with the plasma membrane, thereby delivering membranes and proteins. The mechanism by which secretory vesicles are delivered to the hyphal tip is not clear. In analogy to other polarised cells, such as animal neurons, it is assumed that microtubules and kinesin motors mediate long-distance transport of secretory vesicles, whereas short range motility at the plasma membrane is mediated by another system, consisting of myosins and filamentous actin (F-actin). Indeed, recent studies on numerous filamentous fungi, including the plant pathogen Ustilago maydis, demonstrated that microtubules, kinesins, F-actin and myosins are essential for hyphal growth. However, most conclusions from these data are speculative. This due to the fact that motility of secretory cargo was never visualised and no systematic studies on the whole repertoire of motors in a cell have been undertaken. In this project we will address this challenge. We have developed a microscopic setup that allows us to visualise the motility of individual chitin synthase-containing vesicles. We will label numerous other secretory proteins, including secreted enzymes, and will monitor their delivery to the growing hyphal tip. In co-localisation studies using red and green fluorescent proteins we will determine whether cargo travels in the same or different transport vesicles, thereby elucidating the pathways of secretion. We will determine the cytoskeletal elements that underlie vesicle motility and address the role of all kinesin and myosin motors in secretion by making use of existing mutant protein constructs. Finally, we will further investigate the reason for the bi-directional motility of chitosomes. This behaviour is unexpected and we will investigate whether it is a general feature of secretory vesicles. Subsequently, we will use photoactivatable fluorescent proteins to characterise this motility in order to get an insight into the reason for this phenomenon. In summary, we will combine molecular genetics and life cell imaging to: (1) determine the pathways of secretion in the hyphal cell, (2) address the role of 10 kinesins and 4 myosins in motility of secretory vesicles, (3) characterise the bi-directional motility of secretory vesicles in order to get to an understanding the logic behind this behaviour. The expected outcome of this project will be novel insights into the secretory pathway in filamentous fungi. We will provide a comprehensive understanding of the pathways by which cargo reaches the hyphal tip and of the role of kinesins and myosins in delivery of secretory vesicles. If it is discovered that bi-directional motility of secretory vesicles is a general feature, the current paradigm for secretion will have to be modified. This project will therefore be of fundamental interest to all aspects of fungal research, but it will be of particular importance in understanding fungal pathogenicity and industrial production of recombinant proteins.